Fig 1.
Microscopic images of amastigote-to-trypomastigote transformation.
Amastigotes (1 × 106 cells/mL) were cultured in 80% RPMI-1640 plus 20% Grace’s Insect Medium for 1 day (A) or 4 days (B). Representative microscopic images are shown by using an inverted light compound microscope (Olympus, IX71, Tokyo, Japan) with 60x objective. A video of the same field is available in S1 Movie.
Fig 2.
Optimization of the medium for amastigote-to-trypomastigote transformation.
(A) Amastigotes (1 × 106 cells/mL) were cultured in Grace’s Insect Medium, RPMI-1640 medium, 80% RPMI-1640 plus 20% Grace’s insect medium, or 70% RPMI-1640 plus 20% Grace’s insect medium containing 10% FBS for 7 days. The histogram depicts the percentage of intermediate forms and trypomastigotes, which was calculated by daily counting of the different forms by using IX71 with 60 x objective. More than 100 parasites were randomly counted. (B) The percentage of trypomastigotes is shown in Fig 2A. Data shown are the mean ± S.D. of 3 independent experiments. *P < 0.05, **P < 0.01.
Fig 3.
Biological properties of trypomastigotes derived from in vitro trypomastigogenesis.
(A) Tissue-culture trypomastigotes (a), metacyclic trypomastigotes (b), amastigotes (c), or the parasites cultured for 6 d in trypomastigogenesis medium (d) were fixed, incubated with anti-trans-sialidase antibody, and stained with Alexa Fluor 488-labelled (green) secondary antibody. The nuclei (n) and kinetoplast (k) (blue) were counter-stained using Hoechst 33342. (B) Trypomastigotes derived from trypomastigogenesis or tissue-culture trypomastigotes (2 × 105) were incubated with 2 × 104 3T3-Swiss albino cells for 12 h. For calculation of the infectivity, the number of intracellular parasites in a total of 200 cells was counted after Giemsa staining. Data shown are the mean ± S.D. of 3 independent experiments. Statistical analysis between the groups was performed using Student’s t-test.
Fig 4.
Involvement of TcIP3R in amastigote-to-trypomastigote transformation.
(A) Amastigotes of WT or TcIP3R-SKO- or EGFP-TcIP3R-overexpressing parasites were cultured in a medium composed of 70% RPMI-1640 and 20% Grace’s Insect Medium supplemented with 10% FCS for 7 days. The percentage of the intermediate forms plus trypomastigotes was calculated daily. (B) The percentage of trypomastigotes is shown in Fig 3A. Data shown are the mean ± S.D. of 3 independent experiments. *P < 0.05, **P < 0.01.